at Rutgers University on February 12, 2016

Structural Basis of

O

-Alkylguanine Recognition by a

Bacterial Alkyltransferase-like DNA Repair Protein

Received for publication, December 9, 2009, and in revised form, March 2, 2010 Published, JBC Papers in Press, March 8, 2010, DOI 10.1074/jbc.M109.093591

James M. Aramini‡1, Julie L. Tubbs§, Sreenivas Kanugula¶, Paolo Rossi‡, Asli Ertekin‡, Melissa Maglaqui‡,

Keith Hamilton‡, Colleen T. Ciccosanti‡, Mei Jiang‡, Rong Xiao‡, Ta-Tsen Soong储, Burkhard Rost储, Thomas B. Acton‡,

John K. Everett‡_{, Anthony E. Pegg}¶_{, John A. Tainer}§_{**, and Gaetano T. Montelione}‡ ‡‡2

From the‡Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854 and the Northeast Structural Genomics Consortium, the

§_{Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California}

92037, the¶Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, the储Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032 and the Northeast Structural Genomics Consortium, the**Life Sciences Division, Bioenergy and Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and the‡‡Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

Alkyltransferase-like proteins (ATLs) are a novel class of

DNA repair proteins related to O6_{-alkylguanine-DNA}

alkyl-transferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair pathway. Here, we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus(vpAtl). We demonstrate that vpAtl adopts an AGT-like fold and that the protein is capable of tightly

binding toO6-methylguanine-containing DNA and disrupting

its repair by human AGT, a hallmark of ATLs. Mutation of

highly conserved residues Tyr23_{and Arg}37_{demonstrate their}

critical roles in a conserved mechanism of ATL binding to alkyl-ated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair.

O6_{-Alkylguanine-DNA alkyltransferases (AGTs)}3_{are a large}

family (Pfam, PF01035; EC 2.1.1.63) of alkyl damage-response proteins that reverse endogenous and exogenous alkylation at the O6position of guanines, cytotoxic lesions that otherwise cause G:C to A:T mutations in DNA (1). Human AGT, also called MGMT, interferes with alkylating chemotherapies mak-ing it a target for anticancer drug design (1, 2). AGTs are ubiq-uitous suicide enzymes that mediate the irreversible transfer of the alkyl group to a reactive cysteine within a highly conserved

PCHRV active site sequence motif by a direct reversal mecha-nism, featuring sequence-independent minor-groove binding to a helix-turn-helix motif and flipping of the damaged nucle-otide (3, 4).

Alkyltransferase-like proteins (ATLs), thus far identified in prokaryotes and lower eukaryotes, constitute a new subclass with sequence similarity to AGTs but lacking the critical cys-teine alkyl receptor, which is most often replaced by a trypto-phan (5, 6). ATLs tightly bind a wide range ofO6_{-alkylguanine}

adducts and block the repair ofO6-mG by human AGT but exhibit no alkyltransferase, glycosylase, or endonuclease activ-ities (7–9). The recent first structural study of an ATL (10), from the fission yeast Schizosaccharomyces pombe (spAtl1) which lacks an AGT, provides strong evidence for a novel mechanism of DNA repair in which an ATL binds alkylated DNA in a manner analogous to AGTs, and the resulting non-enzymatic ATL䡠DNA complex triggers the NER pathway (10, 11).

Here, we present the solution NMR structure of the 100-residue ATL fromVibrio parahaemolyticusAQ3810 (Swiss-Prot entry A6B4U8_VIBPA; Northeast Structural Genomics code, VpR247; hereafter referred to as vpAtl), whose structure was solved as part of the Northeast Structural Genomics con-sortium of the National Institutes of Health, NIGMS, Protein Structure Initiative. The vpAtl protein shares 47% sequence identity with spAtl1 and features a PWFRV active site sequence motif (Fig. 1A). We demonstrate that the structure of vpAtl is highly analogous to the AGT fold and that this bacterial protein is capable of tightly binding to O6

-mG-containing DNA and disrupting its repair by human AGT. Site-directed mutagenesis experiments demonstrate the importance of highly conserved residues Tyr23_{and Arg}37_for

binding to alkylated DNA. In addition, the NMR data further suggest that theO6_{-mG recognition cavity of bacterial ATLs}

exhibits some conformational flexibility, which may confer broader specificity for various alkyl guanine lesions. To our knowledge, this work represents the first structural charac-terization of a bacterial ATL.

*This work was supported, in whole or in part, by National Institutes of Health Grant U54-GM074958 (to G. T. M.) from NIGMS (Protein Structure Initiative) and Grants CA097209 (to J. A. T. and A. E. P.) and CA018137 (to S. K.). □S

The on-line version of this article (available at http://www.jbc.org) contains supplemental “Materials and Methods,” Figs. S1–S6, and additional references.

1_{To whom correspondence may be addressed. E-mail: jma@cabm.rutgers.}

edu.

2_{To whom correspondence may be addressed. E-mail: guy@cabm.rutgers.}

edu.

3_{The abbreviations used are: AGT,O}6_{-alkylguanine-DNA alkyltransferase;}

ATL, alkyltransferase-like protein; MES, 2-(N-morpholino)ethanesulfonic acid; NER, nucleotide excision repair; NOE, nuclear Overhauser effect; r.m.s.d., root mean square deviation;O6_-mG,O6_{-methylguanine; hAGT,}

human AGT.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 18, pp. 13736 –13741, April 30, 2010 Printed in the U.S.A.

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EXPERIMENTAL PROCEDURES

Complete details of the methods used in this work are pro-vided in thesupplemental material.

Sample Preparation—The cloning, expression, and purifica-tion of isotopically enriched protein samples of a 100-residue construct from the A79_1377 gene of V. parahaemolyticus AQ3810 (Northeast Structural Genomics code, VpR247; vpAtl) plus C-terminal affinity tag (LEHHHHHH) was performed following standard protocols of the Northeast Structural Geno-mics consortium (12). Samples of [U-13_C,15_{N]- and} [U(5%)-13_{C, (100%)-}15_{N]vpAtl for NMR spectroscopy were}

concen-trated by ultracentrifugation to 0.90 to 0.94 mMin 95% H2O, 5% 2

H2O solution containing 20 mMMES, 200 mMNaCl, 10 mM

dithiothreitol, 5 mM CaCl₂(pH 6.5). Analytical gel filtration

chromatography, static light scattering (supplemental Fig. S1), and one-dimensional15_NT

1andT2relaxation data (

supple-mental Fig. S2) demonstrate that the protein is monomeric in solution under the conditions used in the NMR studies. Single residue mutations of vpAtl (Y23A, Y23F, R37A, and W54A) were cloned using the QuikChange site-directed mutagenesis

kit (Stratagene) and expressed and purified following the same proto-cols used for the wild type protein.

NMR Spectroscopy and Reso-nance Assignment—All NMR spec-tra were collected at 25 °C on Bruker AVANCE 600- and 800-MHz spec-trometers equipped with 1.7-mm TCI and 5-mm TXI cryoprobes, respectively, and a Varian INOVA 600-MHz instrument with a 5-mm HCN cold probe, and referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonic acid. Complete1_H,13_C,

and15_{N resonance assignments for}

vpAtl were determined using con-ventional triple resonance NMR methods. Backbone assignments were made by combined use of AutoAssign 2.4.0 (13) and the PINE 1.0 server (14), and side chain assignment was completed manu-ally. Histidine tautomeric states were elucidated by two-dimen-sional1_H-15_{N heteronuclear}

multi-ple-quantum coherence spectros-copy (15). Resonance assignments were validated using the Assign-ment Validation Suite software package (16) and deposited in the BioMagResDB (accession number 16272).

Structure Determination and Validation—The solution NMR structure of vpAtl was calculated using CYANA 3.0 (17, 18), and the 20 structures with lowest target function out of 100 in the final cycle calculated were further refined by restrained molecular dynam-ics in explicit water with CNS 1.2 (19, 20). Structural statistdynam-ics and global structure quality factors were computed using the PSVS 1.3 software package (21) and MolProbity 3.15 server (22). The global goodness-of-fit of the final structure ensem-bles with the nuclear Overhauser effect spectroscopy (NOESY) peak list data were determined using the RPF anal-ysis program (23). The final refined ensemble of 20 struc-tures (excluding the C-terminal His6) was deposited in the

Protein Data Bank (code 2KIF). All structure figures were made using PyMOL.

15_{N Relaxation Measurements—Residue-specific}

longitudi-nal and transverse15_{N relaxation rates (R}

1andR2) as well as 1_H-15_{N heteronuclear NOE values were obtained on} [U(5%)-13

C, (100%)-15N]vpAtl at a15N Larmor frequency of 60.8 MHz using standard two-dimensional gradient experiments (24). Generalized order parameters,S2_{, were computed from the}

backbone15_{N relaxation and}1_H-15_{N heteronuclear NOE data}

using the Modelfree 4.20 program (25, 26) assuming an isotro-pic model for molecular motion.

FIGURE 1.Solution NMR structure of vpAtl.A,structure-based sequence alignment of vpAtl, spAtl1, and hAGT (residues 91–176). The sequence numbering for vpAtl and the secondary structural elements found in its solution NMR structure are shownabovethe alignment. Identical residues are shown inred. Residues in the helix-turn-helix and active site sequence motifs areboxedinblueandyellow, respectively. Key highly con-served, functionally important residues (Tyr23_{, Arg}37_{, and Trp}54_{in vpAtl) are denoted bytriangles belowthe} alignment.B,stereoview into the putative alkyl-binding site in the lowest energy (CNS) conformer from the final solution NMR structure ensemble of vpAtl. The␣-helices and␤-strands are shown incyanandmagenta, respectively. Side chains of Tyr23_{, Arg}37_{, and Trp}54_{are shown inred, blue,andyellow, respectively.C,ConSurf} (28) image showing the conserved residues in the alkyl-binding site of vpAtl (same view as inB). Residue coloring ranges frommagenta(highly conserved) tocyan(variable) and reflects the degree of residue conser-vation across ATL sequences extracted from the entireO6_{-alkylguanine-DNA methyltransferase protein} domain family (PF01035, Pfam 23.0).D,DelPhi (29) electrostatic surface potential of vpAtl showing negative (red), neutral (white), and positive (blue) charges.

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Human AGT Competition Assays—The inhibition of human AGT activity by spAtl1, vpAtl, and mutants of vpAtl was mea-sured by adding purified hAGT to a preformed mixture of

3_{H-methylated DNA and ATL and then assaying the mixture}

for alkyltransferase activity by determining the transfer of [3_{H]methyl groups from O}6_-[3_{H]methylguanine in DNA to}

purified human AGT protein (27). The assay mixture (1.0 ml), incubated at 37 °C for 15 min, contained 50 mMTris-HCl (pH

7.6), 5 mMdithiothreitol, 50␮g of hemocyanin, 0.1 mMEDTA,

15 ␮g of 3_{H-methylated calf thymus DNA, and different}

amounts of purified ATLs. Ten microliters of purified hAGT (0.5 pmol) were added to the above reaction mixture, and incu-bation was continued for 60 min at 37 °C and assayed for alkyl-transferase activity.

RESULTS

Solution NMR Structure of vpAtl—The structure of vpAtl adopts an AGT-like fold composed of five ␣-helices (␣1, Asp2_–His13_{; ␣2, Tyr}23_–Gly31_{; ␣3, Tyr}35_–Leu46_{; ␣4, Gly}68_–

Ala80_{;␣5, Ala}92_–Lys97_{) and two short antiparallel␤-strands}

(␤1, Ser21–Thr22;␤2, Val57–Ile58) in the core of the protein (Fig. 1B; supplemental Fig. S3). Structural statistics and a summary of the NMR data from this study are provided in Table 1 andsupplemental Fig. S4, respectively. By analogy to homologs (see below), helices 2 and 3 include the helix-turn-helix DNA-binding motif. A ConSurf (28) analysis of all ATLs from the AGT protein domain family (PF01035) reveals that highly conserved residues, demonstrated in spAtl1 (10) to be involved in flipping of the damaged alkyl guanine (Tyr23 _{and Arg}37_{), interacting with the orphaned}

cytosine (Arg37_{), and binding to the alkyl moiety (Tyr}23_and

Trp54_{), are clustered in a partially occluded binding pocket}

(Fig. 1C). This face of the protein also features a quite posi-tive electrostatic surface potential (29) due to several basic residues, consistent with its role in DNA binding (Fig. 1D;

supplemental Fig. S5). The binding pocket is flanked by the less conserved binding site loop (preceding helix 4) and C-terminal loop (between helices 4 and 5). In spAtl1 these loops are important for open-to-closed (free-to-bound) con-formational changes and mediate interactions with proteins in the NER pathway, respectively (10). Two histidines (His13

in helix 1 and His38_{flanking Arg}37_{in helix 3), largely unique}

to ATLs fromVibriospecies, adopt neutral N⑀2_{H tautomers}

under the conditions used in this study (supplemental Fig. S6).

Backbone Dynamics of vpAtl—The internal dynamic prop-erties of vpAtl were further investigated using backbone15_N

relaxation and heteronuclear NOE experiments (24). Reduced15_NR

2relaxation rates and

1_H-15_{N heteronuclear}

NOE values are observed for residues Leu46_{to Leu}52_{, which}

span the C-terminal end of helix 3 and the loop preceding the heart of the substrate-binding site (Fig. 2A). These data result in reduced order parameters, S2, indicative of enhanced backbone motions within this binding pocket cap. Moreover, there are a handful of weak/missing backbone amide NMR resonances in the protein, consistent with con-formational exchange broadening. Mapping these effects onto the structure of vpAtl clearly reveals that several

con-formationally dynamic residues cluster around the substrate binding pocket (Fig. 2B). This plasticity provides a structural basis for the broad range of guanine lesions that can be rec-ognized by ATLs (8, 11); flexibility in the recognition cavity provides a capacity for molding the binding site around var-ious alkyl guanine lesions besidesO6_-mG.

TABLE 1

Summary of NMR and structural statistics for vpAtl

Structural statistics were computed for the ensemble of 20 deposited structures (PDB entry, 2KIF). r.m.s., root mean square; r.m.s.d., r.m.s. deviation.

Completeness of resonance assignmentsa

Backbone 97.6%

Side chain 98.3%

Aromatic 100%

Stereospecific methyl 100% Conformationally restricting constraintsb

Distance constraints Total 2448 Intra-residue (i⫽j) 621 Sequential (兩i⫺j兩⫽1) 547 Medium range (1⬍兩i⫺j兩⬍5) 493 Long range (兩i⫺j兩ⱖ5) 787 Distance constraints/residue 24.2 Dihedral angle constraints 125 Hydrogen bond constraints

Total 62

Long range (兩i⫺j兩ⱖ5) 8 No. of constraints/residue 26.1 No. of long range constraints/residue 7.9 Residual constraint violationsb

Average no. of distance violations/structure

0.1–0.2 Å 1.55

0.2–0.5 Å 0.1

⬎0.5 Å 0

Average r.m.s. distance violation/constraint 0.01 Å Maximum distance violation 0.31 Å Average no. of dihedral angle violations/structure

1–10° 1.7

⬎10° 0

Average r.m.s. dihedral angle violation/constraint 0.37° Maximum dihedral angle violation 4.60° r.m.s.d. from average coordinatesb,c

Backbone atoms 0.5 Å

Heavy atoms 0.8 Å

Procheck Ramachandran statisticsb,c

Most favored regions 91.5% Additional allowed regions 8.5% Generously allowed 0.0% Disallowed regions 0.0% MolProbity Ramachandran statisticsd

Favored regions 95.4%

Allowed 100.0%

Global quality scoresb

Raw Z-score Verify3D 0.48 0.32 ProsaII 0.91 1.08 Procheck(␾-␺)c _{⫺0.13 ⫺0.20} Procheck(all)c _{⫺0.02 ⫺0.12} Molprobity clash 18.92 ⫺1.72 RPF scorese Recall 0.974 Precision 0.937 Fmeasure 0.955 DP score 0.835 a

Computed using AVS software (16) from the expected number of peaks, excluding the following: highly exchangeable protons (N-terminal, Lys, and Arg amino groups, hydroxyls of Ser, Thr, and Tyr), carboxyls of Asp and Glu, nonprotonated aromatic carbons, and the C-terminal His6tag.

b

Calculated using PSVS 1.3 program (21). Average distance violations were calcu-lated using the sum overr⫺6

. c

Ordered residue ranges (S(␾)⫹S(␺)⬎1.8): 2–32, 35– 47, 50 –100. d

Calculated for all residues in the ensemble using the MolProbity 3.15 server (22). e

RPF scores (23) reflecting the goodness-of-fit of the final ensemble of structures (including disordered residues) to the NMR data.

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Comparison to Related Structures—The solution structure of vpAtl is structurally very similar to the recent crystal structures of bound and free spAtl1 (Dali (30) Z-scores: 3GYH, 16.1; 3GX4, 15.7; 3GVA, 15.0; C␣r.m.s.d. values: 3GYH, 1.7 Å; 3GX4, 1.8 Å; 3GVA, 1.9 Å) (10) as well as to structures of the C-termi-nal domains of human AGT (3, 4, 31, 32), Escherichia coli Ada-C (33), and archaeal (34, 35) AGTs (DaliZ-scores ranging from 5.4 to 11.1), which share⬍30% sequence identity with vpAtl. Considering the metric of modeling leverage (36), an important measure of the new structural information provided by a protein structure, the vpAtl structure has a novel leverage value of 26 models and total modeling leverage value of 1,493 structural models (UniProt release 12.8; PSI BlastE⬍10⫺10_).

Of these, 17 sequences are putative ATLs from eukaryotes, including the recently identified ATL from sea anenome (10). The vpAtl structure most closely resembles the closed (DNA-bound) form of spAtl1, and conserved residues important for damage recognition and binding superimpose well in the struc-tures (Fig. 3A). There are, however, subtle differences between the structures. Both the binding site and C-terminal loops are shorter in vpAtl, and the binding pocket in vpAtl is partially buried by residues from the binding site loop (Ser65_{and Leu}66₎

and the binding pocket cap (Leu46–Pro53), resulting in a much smaller binding pocket (⬍Area⬎(Å2_{)⫽185⫾61) than that}

observed for spAtl1 (10). However, as discussed above, the15_N

relaxation and 1_H-15_{N heteronuclear NOE data along with}

weak/missing backbone amide resonances indicate that there are backbone conformational dynamics within the binding

pocket cap (Fig. 2), suggesting that vpAtl may adopt conforma-tions in which the substrate binding pocket is more exposed.

Inhibition of hAGT-mediated DNA Repair by vpAtl—Like the ATL proteins fromE. coli, S. pombe,andThermus ther-mophilus(7–9), vpAtl does not exhibit alkyltransferase activity (data not shown). However, a common trait of ATLs is their ability to tightly bindO6-alkylguanine DNA, with up to sub-nanomolar affinity (10), and prevent its repair by AGTs. The inhibition of human AGTO6_{-mG repair by vpAtl and spAtl1 is}

shown in Fig. 3B. In this assay, varying amounts of ATL are preincubated with3_{H-methylated DNA, followed by}

incuba-tion with human AGT and measurement of the amount of radiolabel transferred to the AGT (27). As expected for an ATL, vpAtl strongly inhibitsO6_{-mG repair by hAGT when present in}

molar excess and exhibits a similar affinity for alkylated DNA compared with spAtl1.

The DNA binding roles of selected conserved residues in vpAtl, namely Tyr23_{, Arg}37_{, and Trp}54_{, were further examined}

using this competition assay (Fig. 3,CandD). Replacing either Tyr23_{or Arg}37_{with alanine yields mutants that have no effect}

on hAGT activity, meaning that their ability to bind alkylated DNA is severely impaired. On the other hand, the Y23F mutant is still capable of blocking repair of methylated DNA by hAGT, albeit not as effectively as wild type vpAtl (Fig. 3C). These data are consistent with the requirement of a bulky aromatic residue at the position of Tyr23_{to flip the damaged guanine base into}

the binding pocket and the function of Arg37_{to intercalate the}

DNA and hydrogen bond to the orphaned cytosine (10). Similar

FIGURE 2.Backbone dynamics of vpAtl.A,plots of backbone amide15_NR

1andR2relaxation rates,

1_H-15_{N heteronuclear NOEs, and generalized order} parameters,S2_{,versusresidue number obtained on [U(5%)-}13_{C, (100%)-}15_{N]-vpAtl at a}15_{N Larmor frequency of 60.8 MHz. Order parameters were computed} using the Modelfree 4.20 program (25, 26) assuming an isotropic model, yielding an overall rotational correlation time,␶c, of 7.9 ns.B,backbone dynamics of

vpAtl mapped onto its structure. Residues withS2_{ⱕ0.7, indicative of enhanced backbone flexibility, are inred, and residues with weak or missing}1_H-15_N heteronuclear single quantum coherence resonances are coloredyellow. Prolines are shown ingray, and the substrate binding pocket and binding pocket cap are indicated.

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effects were observed in analogous mutagenesis experiments on hAGT (37, 38). Finally, swapping Trp54_{for an alanine (Trp}

and Ala are present in⬇89 and ⬇9%, respectively, of ATLs; Pfam 23.0) results in an ATL that also exhibits some affinity for methylated DNA, although less than that for wild type vpAtl (Fig. 3D); a similar effect was observed for the ATL fromE. coli (7). Overall, the relative affinities of the ATLs and mutants studied here for O6_{-mG DNA follow the trend: spAtl1 ⱖ}

vpAtl⬎[Y23F]-vpAtl ⬎[W54A]-vpAtl⬎⬎[Y23A]-vpAtl⬇ [R37A]-vpAtl.

DISCUSSION

The results on vpAtl presented here demonstrate a high degree of structural conservation between bacterial and yeast ATLs and demonstrate that vpAtl exhibits the hallmark bio-chemical behavior of ATLs. Furthermore, mutation of highly conserved residues Tyr23_{or Arg}37_{to alanine abolishes the}

abil-ity of vpAtl to block methylated DNA repair by human AGT, and the affinity of the Y23F mutant for methylated DNA dem-onstrates the necessity of a bulky aromatic residue in this posi-tion. To our knowledge, this is the first mutagenesis study examining these critical residues in ATLs and their roles in binding toO6_{-mG DNA and blocking human AGT activity.}

Taken together, our structural and mutagenesis results provide strong evidence for a conserved mecha-nism of ATL binding to alkylated DNA mediated by critical tyrosine and arginine residues and involving the flipping out of the damaged base (10).

Like several other organisms, includingE. coli,V. parahaemolyti-cus possesses genes for both ATL and AGT. It was recently shown that repair of O6-alkylguanine lesions in E. coli is segregated between the direct repair (AGT) and ATL-coupled NER pathways on the basis of the size of the alkyl group, with the latter repairing O6_{-alkylguanine adducts larger in}

size than a methyl group, which are poor substrates for bacterial AGTs (11). Moreover, the high structural similarity between free vpAtl and free and bound spAtl1 suggests that vpAtl also mediates O6

-alkylgua-nine repair by recruitment of pro-teins involved in NER. Hence, we postulate that vpAtl is also capable of interacting with a broad range of O6_{-alkylguanine substrates and}

likely mediates an analogous cross-talk between alkyltransferase and NER repair pathways. In this sce-nario, vpAtl would function to channel bulkier O6_{-alkylguanine}

lesions into the NER pathway. Con-formational dynamics within the recognition cavity of apo-ATL, revealed for the first time by this structural NMR study, confer functional plasticity that may be essential for providing its wider range of guanine lesion specificity.

In light of the recent structural and biochemical character-ization ofS. pombeAtl1 and the predicted occurrence of ATLs in archaea (10), our results for vpAtl also provide further sup-port for the hypothesis that ATLs are an ancient class of non-enzymatic proteins at the interface of the base and nucleotide excision repair pathways for DNA repair. These results thus help provide a unified understanding of DNA damage responses, which has been a major goal for the structural biol-ogy of DNA repair since the discovery of base and nucleotide excision repair pathways (39).

Acknowledgments—We thank R. L. Belote, G. V. T. Swapna, and M. Fischer for valuable scientific discussions.

REFERENCES

1. Tubbs, J. L., Pegg, A. E., and Tainer, J. A. (2007)DNA Repair6,1100 –1115 2. Verbeek, B., Southgate, T. D., Gilham, D. E., and Margison, G. P. (2008)Br.

Med. Bull.85,17–33

FIGURE 3.Conserved structure and DNA binding properties of vpAtl.A,overlay of the vpAtl structure (cyan) and the crystal structure of spAtl1 bound toO6_{-mG-DNA (Protein Data Bank code 3GX4;magenta) (10). The side} chains of Tyr23_{, Arg}37_{, and Trp}54_{are shown inred, blue,andyellow, respectively, and theO}6_{-mG in the bound} spAtl1 structure is shown ingray.B,percent activity of hAGT as a function of ATL concentration for vpAtl (triangles) and spAtl1 (circles).C,effect of mutating Tyr23_{in vpAtl on hAGT activity; wild type vpAtl (black),} [Y23A]-vpAtl (red triangles), and [Y23F]-vpAtl (red circles).D,effect of mutating Arg37_{and Trp}54_{in vpAtl on hAGT} activity; wild type vpAtl (black), [R37A]-vpAtl (blue), and [W54A]-vpAtl (gold).

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Structure of a Bacterial Alkyltransferase-like Protein

APRIL 30, 2010 •VOLUME 285 • NUMBER 18 JOURNAL OF BIOLOGICAL CHEMISTRY 13741

at Rutgers University on February 12, 2016

http://www.jbc.org/

SUPPLEMENTARY INFORMATION FOR:

Structural Basis of O6_{-Alkylguanine Recognition by a Bacterial Alkyltransferase-like DNA} Repair Protein

James M. Aramini‡, Julie L. Tubbs§, Sreenivas Kanugula¶_{, Paolo Rossi}‡

, Asli Ertekin,‡ Melissa Maglaqui‡, Keith Hamilton‡, Colleen T. Ciccosanti‡, Mei Jiang‡, Rong Xiao‡, Ta-Tsen Soong||, Burkhard Rost||, Thomas B. Acton‡, John K. Everett‡, Anthony E. Pegg¶, John A. Tainer§,† and

Gaetano T. Montelione‡,⊥

From the ‡Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854

and Northeast Structural Genomics Consortium, §Skaggs Institute for Chemical Biology and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037,

¶_{Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center,}

Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, ||Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032

and Northeast Structural Genomics Consortium, †Life Sciences Division, Bioenergy and Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and

⊥_{Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine} and Dentistry of New Jersey, Piscataway, New Jersey 08854

Materials and Methods

The 100-residue construct from the A79_1377 gene of Vibrio parahaemolyticus AQ3810 (NESG ID, VpR247; hereafter referred to as vpAtl) was cloned into a pET21 expression vector (Novagen) containing a C-terminal affinity tag (LEHHHHHH), yielding the plasmid VpR247-21.9. The VpR247-21.9 plasmid was transformed into codon enhanced BL21 (DE3) pMGK

Escherichia coli cells, and cultured in MJ9 minimal medium1 containing (15NH4)2SO4 and U -13_{C-glucose as the sole nitrogen and carbon sources. Initial cell growth was carried out at 37}o_C

and protein expression was induced at 17oC by 1 mM isopropyl-β-D-thiogalactopyranoside

(IPTG). Expressed proteins were purified using an ÄKTAxpress™ (GE Healthcare) two-step protocol consisting of HisTrap HP affinity chromatography followed directly by HiLoad 26/60 Superdex 75 gel filtration chromatography. The final yield of purified isotopically-enriched vpAtl was ≈ 18 mg/L of culture. Samples of [U-13C,15N]- and [U-5%-13C,100%-15N]-vpAtl for NMR spectroscopy were concentrated by ultracentrifugation to 0.90 to 0.94 mM in 95% H2O / 5% 2_H

2O solution containing 20 mM MES, 200 mM NaCl, 10 mM DTT, 5 mM CaCl2 at pH 6.5. Sample purity and molecular mass were confirmed by SDS-PAGE and MALDI-TOF mass spectrometry (MALDI-TOF mass of [U-13C,15N]-vpAtl (Da): experimental, 13,038.6; expected, 13,038). Analytical gel filtration chromatography, static light scattering and 15N T1 and T2 relaxation data demonstrate that the protein is monomeric in solution under the conditions used in the NMR studies. Single residue mutations of vpAtl (Y23A, Y23F, R37A, and W54A) were cloned using the QuikChange site-directed mutagenesis kit (Stratagene), and expressed and purified following the same protocols used for the wild type protein.

All NMR data were collected at 25o_{C on Bruker AVANCE 600 and 800 MHz} spectrometers equipped with 1.7-mm TCI and 5-mm TXI cryoprobes, respectively, and a Varian INOVA 600 MHz instrument with a 5-mm HCN cold probe, processed with NMRPipe2 and

visualized using SPARKY.3 Complete 1H, 13C, and 15N resonance assignments for vpAtl were determined using conventional triple resonance NMR methods and deposited in the BioMagResDB (BMRB accession number 16272). All spectra were referenced to internal DSS. Backbone assignments were made by combined use of AutoAssign 2.4.04 and the PINE 1.0 server5 _{using peak lists from 2D} 1_H-15_{N HSQC and 3D HNCO, HN(CA)CO, HN(CO)CA,} HNCA, CBCA(CO)NH and HNCACB spectra. Side chain assignment was completed manually using 3D HBHA(CACO)NH, HCCH-COSY, HCCH-TOCSY and (H)CCH-TOCSY experiments. Stereospecific isopropyl methyl assignments for all Val and Leu residues were deduced from characteristic cross-peak fine structures in high resolution 2D 1H-13C HSQC spectra of [U-5%-13_C,100%-15_N]-vpAtl.6 _{Resonance assignments were validated using the} Assignment Validation Suite (AVS) software package.7 Three-bond 3J(HN-Hα_{) scalar couplings}

were determined using the 3D HNHA experiment.8 1H-15N heteronuclear NOE and 15N T1 and

T2 relaxation measurements were made using gradient sensitivity-enhanced 2D heteronuclear NOE and 1D and 2D 15_{N T}

1 and T2 (CPMG) relaxation experiments, respectively.9 The tautomeric states of histidines were elucidated by 2D 1H-15N heteronuclear multiple-quantum coherence (HMQC) spectra.10

The solution NMR structure of vpAtl was calculated using CYANA 3.011,12 supplied with peak intensities from 3D simultaneous CN NOESY13 (τm = 100 ms) and 3D 13C-edited aromatic NOESY (τm = 120 ms) spectra, together with broad dihedral angle constraints computed by TALOS14 (φ ± 30°; ψ ± 30°). The 20 structures with lowest target function out of 100 in the

final cycle calculated were further refined by restrained molecular dynamics in explicit water using CNS 1.215,16 and the PARAM19 force field, using the final NOE derived distance constraints, TALOS dihedral angle constraints and hydrogen bond constraints derived from AutoStructure 2.2.117 _{and CYANA. In this final stage of the structure determination rotamer}

states of specific ordered residues were constrained (χ1, χ2 ± 20°) based on Molprobity server18 and PROCHECK19 analyses and neutral histidine Nε2_{H tautomers were defined at H13 and H38.}

The final refined ensemble of 20 structures (excluding the C-terminal His6) was deposited into the Protein Data Bank (PDB ID, 2KIF). Structural statistics and global structure quality factors, including Verify3D,20 ProsaII,21 PROCHECK,19 and MolProbity18,22 raw and statistical Z-scores, were computed using the PSVS 1.3 software package.23 The global goodness-of-fit of the final structure ensembles with the NOESY peak list data were determined using the RPF analysis program.24 _{Structure based sequence alignments and coordinate superimpositions were obtained} from the CE combinatorial extension server.25 Three-dimensional protein structure comparison of the vpAtl structure with structures in the Protein Data Bank was conducted using the DaliLite server26. Conserved residue analysis was performed using the ConSurf server27 on ATL sequences extracted from the entire O6-alkylguanine-DNA methyltransferase protein domain family (PF01035, Pfam 23.0, 245 sequences out of 1426 sequences), and re-aligned with Clustal X 2.0.28 The size of the binding pocket (average value over the ensemble with hydrogens stripped away by the MolProbity server18) was obtained from the CASTp server29 using a 1.1 Å probe radius. All structure figures were made using PyMOL.30

Residue specific 15N longitudinal and transverse relaxation rates (R1 and R2) and 1H-15N heteronuclear NOE values were calculated from cross-peak intensities in the respective 2D experiments9 obtained on [U-5%-13C,100%-15N]-vpAtl at a 15N Larmor frequency of 60.8 MHz using in-house written codes in MATLAB 7.9.0 (MathWorks). T1 spectra were acquired with delays, T = 10, 50, 100, 150, 250, 500, 750, 1000, 1500, and 2000 ms, and a relaxation delay of 2s. T2 spectra were acquired with CPMG delays, T = 10, 30, 50, 70, 90, 110, 130, 170, and 210 ms, and a relaxation delay of 1.5s. For the 1_H-15_{N heteronuclear NOE measurements, relaxation} delays of 5s and 2s followed by a 3s proton saturation period were used for the reference (no

NOE) and NOE spectra, respectively. In all cases, peak intensities of resolved resonances were analyzed; data for the handful of severely overlapping resonances were omitted from all analyses. Longitudinal and transverse relaxation rates were computed by fitting peak intensity

I(T) as a function of delay time, T, according to Eqs. 1 and 2, respectively:31

!

I(T)=I_"#

_[

_]

_{

_}

!

I(T)=I₀exp

_{

_}

The uncertainties of the intensities were estimated from the root-mean-square baseline noise in the spectra. The statistical properties of the resulting relaxation rates were estimated from randomly generated data sets using a Monte Carlo approach, based on the uncertainties of the spectral intensities.31 _{Generalized order parameters, S}2_{, were computed from the backbone} 15_N relaxation and 1H-15N heteronuclear NOE data using the Modelfree 4.20 program31,32 interfaced with the FASTModelFree program.33 An isotropic model for molecular motion was used, yielding an overall rotational correlation time, τc, of 7.9 ns.

The inhibition of human AGT activity by spAtl1, vpAtl and mutants of vpAtl was measured by adding purified hAGT to a preformed mixture of [3H]-methylated DNA and ATL and then assaying the mixture for alkyltransferase activity. Alkyltransferase activity was measured by determining the transfer of [3H]-methyl groups from O6-[3H]-methylguanine in DNA to purified human AGT protein.34 The assay mixture (1.0 ml), incubated at 37°C for 15 min, contained 50 mM Tris-HCl (pH 7.6), 5 mM DTT, 50 µg hemocyanin, 0.1 mM EDTA, 15 µg of [3H]-methylated calf thymus DNA and different amounts of purified ATLs. Ten microliters of purified human AGT protein (0.5 pmoles) were added to the above reaction mixture, and incubation was continued for 60 min at 37°C and subsequently assayed for alkyltransferase activity.

Supplementary Figure S1. Static light scattering results for vpAtl. Data were collected on a miniDAWN (TREOS) Light Scattering instrument (Wyatt Technology) coupled with an analytical gel filtration column at λ= 690 nm, a flow-rate of 0.5 ml/min and at room temperature

on an NMR sample of [U-5%-13C, 100%-15N]-vpAtl at pH 6.5. Inset: Plot of molar mass versus elution volume. The resulting experimental molecular weight of vpAtl is 13.6 kDa; the expected MW including affinity tag is 12.5 kDa.

Supplementary Figure S2. 1D 15N T1 and T2 relaxation data for [U-5%-13C,100%-15N]-vpAtl. The data were acquired on a Bruker AVANCE 600 MHz spectrometer at 298 K using pseudo-2D 15_{N T}

1 and T2 gradient experiments.9 T1 spectra were acquired with delays, T = 20, 50, 100, 200, 300, 400, 600, 800, 1000, 1200 and 1500 ms, and a relaxation delay of 3s. T2 spectra were acquired with CPMG delays, T = 16, 32, 48, 64, 80, 96, 128, 160, 192, 240 and 320 ms, and with a relaxation delay of 1.5s. (Top): 15N T1 and T2 values were extracted by plotting the decay of integrated 1HN intensity between δ ≈ 8.4 to 9.8 ppm and fitting the curves with standard

exponential equations using the program ‘t1guide’ within Topspin2.1 (Bruker BioSpin). (Bottom): Plot of rotational correlation time, τc (ns), versus protein molecular weight (kDa) for known monomeric NESG targets of ranging size (taking into account isotope enrichment as well as affinity tags in the sequence). 15N T1/T2 data for all monomeric proteins used for the τc vs. MW plot were obtained on the same Bruker 600 MHz spectrometer at 298 K, and analyzed as described above. For each protein, the τc was calculated from the 15N T1/T2 ratio using the following approximation of literature relaxation equations:35

! "c# 6T 1 T2 $7 % & ' ( ) */4+,N (3)

where νN is the resonance frequency of 15N in Hz.

Using this approach, we obtain a τc of 8.0 ns for [U-5%-13C,100%-15N]-vpAtl, shown in blue, which is consistent with a monomer (expected MW = 12.5 kDa, including C-terminal LEHHHHHH affinity tag).

Supplementary Figure S3. Superposition of the final ensemble of 20 conformers representing the solution NMR structure of Vibrio parahaemolyticus ATL, vpAtl (PDB entry, 2KIF). The α

-helices and β-strands are shown in cyan and magenta, respectively, and all secondary structure

Supplementary Figure S4. NMR connectivity map summarizing data used to determine resonance assignments and secondary structure for vpAtl. The final six unassigned histidines in the C-terminal tag have been omitted. Intraresidue (i) and sequential (s) connectivities for the three-rung assignment strategy4 _{matching intraresidue and sequential C}

′, Cα_{, and C}β _resonances

are shown as horizontal red and yellow lines, respectively. 3J(HN-Hα_{) values range as follows:}

(o) < 5.0 Hz; ( ) 5.0 ≤ J≤ 7.5 Hz; (●) > 7.5 Hz. Interresidue NOE connectivities are shown as thin, medium, and thick black lines, corresponding to weak, medium, and strong NOE interactions. Bar graphs of the consensus CSI36 _and 1_H-15_{N heteronuclear NOE data are shown} in blue. The secondary structural elements in the final vpAtl NMR structure (2KIF) are also shown. In general, the secondary structural elements in the final structure are well defined by the 3J(HN-Hα_{) scalar coupling,} 1_H-15_{N heteronuclear NOE and NOESY patterns.}

Supplementary Figure S5. DelPhi37 electrostatic surface potential of vpAtl. (Left) Electrostatic surfaces showing negative (red), neutral (white), and positive (blue) charges. (Right) Same orientation as on the left, showing the secondary structural elements and key residues – Y23, R37 and W54.

Supplementary Figure S6. Top: The 800 MHz 1H-15N HMQC NMR spectrum of [U -5%-13_C,100%-15_{N]-vpAtl in pH 6.5 buffer at 25}o_{C. Acquisition parameters:} 15_{N carrier at 205 ppm,} 2_J(15_N-1_{H) = 22 Hz, 2s relaxation delay, 2048 x 300 complex points, 200 ppm sweep width in the} 15_{N dimension, and 80 transients per t}

1 increment. Assigned cross-peaks for H13 and H38 are labeled. The Γ pattern is indicative of the Nε2_{H neutral histidine tautomer (inset).}10 _{All other}

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John A. Tainer and Gaetano T. Montelione

Ta-Tsen Soong, Burkhard Rost, Thomas B. Acton, John K. Everett, Anthony E. Pegg, Melissa Maglaqui, Keith Hamilton, Colleen T. Ciccosanti, Mei Jiang, Rong Xiao, James M. Aramini, Julie L. Tubbs, Sreenivas Kanugula, Paolo Rossi, Asli Ertekin,

Alkyltransferase-like DNA Repair Protein

-Alkylguanine Recognition by a Bacterial 6

O

Structural Basis of

doi: 10.1074/jbc.M109.093591 originally published online March 8, 2010 2010, 285:13736-13741.

J. Biol. Chem.

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